Thyroid Hormone-disrupting Chemicals Research Articles

Effect of thyroid hormone-disrupting chemicals on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish.

We compared the influence of thyroid hormone-disrupting chemicals (heptafluorobutanoic acid, PFBA and tris[1,3-dichloro-2-propyl] phosphate, TDCPP) and thyroid hormone (3,3',5-triiodo-L-thyronine, T3) on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish. The swim bladder of most larvae had inflated at 4h post hatching (hph) in Japanese medaka and at 48 hph in zebrafish in controls. In both fish species, the swim bladder inflation was inhibited in larvae exposed to PFBA (lowest observed effect concentration [LOEC] in medaka: 40 mg/L; in zebrafish: 80 mg/L), TDCPP (LOEC in medaka: 1mg/L; in zebrafish: 0.5mg/L), and T3 (no inhibition in Japanese medaka; LOEC in zebrafish: 7.5μg/L). We also examined the influence of PFBA, TDCPP, and T3 on the expression of thyroid stimulating hormone subunit beta (tshβ) or thyroid hormone receptor alpha (trα) and beta (trβ). No changes were observed in the expression of genes after PFBA and TDCPP exposure; however, T3 exposure upregulated trα and trβ expression in both fish species. When the results were compared between Japanese medaka and zebrafish, swim bladder inflation in both species was found to be inhibited by exposure to thyroid hormone-disrupting chemicals. Our results show that inhibition of the swim bladder inflation at 4 hph in Japanese medaka and 48 hph in zebrafish is a potential indicator of thyroid hormone-disturbing activity of chemicals.

Homology modeling to screen for potential binding of contaminants to thyroid hormone receptor and transthyretin in glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus)

Thyroid hormone disrupting chemicals (THDCs) are of major concern in ecotoxicology. With the increased number of emerging chemicals on the market there is a need to screen for potential THDCs in a cost-efficient way, and in silico modeling is an alternative to address this issue. In this study homology modeling and docking was used to screen a list of 626 compounds for potential thyroid hormone disrupting properties in two gull species. The tested compounds were known contaminants or emerging contaminants predicted to have the potential to reach the Arctic. Models of transthyretin (TTR) and thyroid hormone receptor α and β (TRα and TRβ) from the Arctic top predator glaucous gull (Larus hyperboreus) and temperate predator herring gull (Larus argentatus) were constructed and used to predict the binding affinity of the compounds to the thyroid hormone (TH) binding sites. The modeling predicted that 28, 4 and 330 of the contaminants would bind to TRα, TRβ and TTR respectively. These compounds were in general halogenated, aromatic and had polar functional groups, like that of THs. However, the predicted binders did not necessarily have all these properties, such as the per- and polyfluoroalkyl substances that are not aromatic and still bind to the proteins.

Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish.

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction (e.g., phosphodiesterase, opsins) were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class “sensory perception” differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes.

Identification of hepatic thyroid hormone-responsive genes in neonatal rats: Potential targets for thyroid hormone-disrupting chemicals

There have been many concerns about the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. Because thyroid hormones are essential for regulating the growth and differentiation of many tissues, disruption of thyroid hormones during the neonatal period of an organism might lead to permanent effects on that organism. We postulated that there are target genes that are sensitive to thyroid hormones particularly during the neonatal period and that would thus be susceptible to thyroid hormone-disrupting chemicals. Global gene expression analysis was used to identify these genes in the liver of rat neonates. The changes in hepatic gene expression were examined 24 h after administering 1.0, 10, and 100 ng/g body weight (bw) triiodothyronine (T3) to male rats on postnatal day 3. Thirteen upregulated and four downregulated genes were identified in the neonatal liver. Among these, Pdp2 and Slc25a25 were found to be upregulated and more sensitive to T3 than the others, whereas Cyp7b1 and Hdc were found to be downregulated even at the lowest dose of 1.0 ng/g bw T3. Interestingly, when the responses of gene expression to T3 were examined in adult rats (8-week old), one-third of them did not respond to T3. The environmental chemicals with thyroid hormone-like activity, hydroxylated polybrominated diphenyl ethers, were then administered to neonatal rats to examine the effects on expression of the identified genes. The results showed that these chemicals were indeed capable of changing the expression of Slc25a25 and Hdc. Our results demonstrated a series of hepatic T3-responsive genes that are more sensitive to hormones during the neonatal period than during adulthood. These genes might be the potential targets of thyroid hormone-disrupting chemicals in newborns.

Altered expression of the Olr59, Ethe1, and Slc10a2 genes in the liver of F344 rats by neonatal thyroid hormone disruption.

Many concerns have been expressed regarding the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. The disruption of thyroid hormones in the neonatal period may lead to permanent effects on thyroid hormone homeostasis as well as related developmental disorders, as thyroid hormones are essential for regulating the growth and differentiation of many tissues. To understand the long-term alteration in gene expressions by neonatal administration of thyroid hormone-like chemicals in general, we identified genes whose expression was altered in the liver, an important component of the thyroid hormone axis, by neonatal exposure to triiodothyronine (T3). T3 was administered to male F344 rats on postnatal days 1, 3, and 5 (week 0). At 8 weeks of age, cDNA microarray analysis was used to identify hepatic genes whose expression was altered by neonatal exposure to T3. Among the up-regulated genes that were identified, the expression of Olr59, Ethe1, and Slc10a2 increased specifically in rats neonatally exposed to T3. Interestingly, altered hepatic expression of these genes indeed increased when a hydroxylated polybrominated diphenyl ether (PBDE), OH-BDE42, which is capable of binding to the TR, was given neonatally. Our data demonstrated that neonatal exposure to thyroid hormones could affect the long-term expression of the genes, which could be useful markers for neonatal effects by thyroid hormone-disrupting chemicals. Copyright © 2017 John Wiley & Sons, Ltd.

Structure-Based Virtual Screening Protocol for in Silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin.

Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 μM. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.

Identification and Molecular Interaction Studies of Thyroid Hormone Receptor Disruptors among Household Dust Contaminants.

Thyroid hormone disrupting chemicals (THDCs), often found abundantly in the environment, interfere with normal thyroid hormone signaling and induce physiological malfunctions, possibly by affecting thyroid hormone receptors (THRs). Indoor dust ingestion is a significant human exposure route of THDCs, raising serious concerns for human health. Here, we developed a virtual screening protocol based on an ensemble of X-ray crystallographic structures of human THRβ1 and the generalized Born solvation model to identify potential THDCs targeting the human THRβ1 isoform. The protocol was applied to virtually screen an in-house indoor dust contaminant inventory, yielding 31 dust contaminants as potential THRβ1 binders. Five predicted binders and one negative control were tested using isothermal titration calorimetry, of which four, i.e., 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether (BADGE-HCl-H2O), 2,2',4,4'-tetrahydroxybenzophenone (BP2), and 2,4-dichlorophenoxyacetic acid (2,4-D), were identified as THRβ1 binders with binding affinities ranging between 60 μM and 460 μM. Molecular dynamics (MD) simulations were employed to examine potential binding modes of these binders and provided a rationale for explaining their specific recognition by THRβ1. The combination of in vitro binding affinity measurements and MD simulations allowed identification of four new potential THR-targeting THDCs that have been found in household dust. We suggest using the developed structure-based virtual screening protocol to identify and prioritize testing of potential THDCs.

Permethrin is a potential thyroid-disrupting chemical: In vivo and in silico envidence

Permethrin (PM), one of the most heavily used synthetic pyrethroids, has the potential to interfere with thyroid hormones in mammals, however, the effect is poorly recognized in aquatic organisms. Herein, embryonic zebrafish were exposed to PM (0, 1, 3 and 10μg/L) until 72h post-fertilization. We demonstrated that PM readily accumulated in larvae with a preference for cis-PM, inhibited development and increased thyroxine and 3,5,3′-triiodothyronine levels accompanying increase in the transcription of most target genes, i.e., thyroid-stimulating hormone β, deiodinases, thyroid receptors, involved in the hypothalamic-pituitary-thyroid axis. Further Western blot analysis indicated that transthyretin (TTR) protein was significantly increased. Molecular docking analysis and molecular dynamics simulations revealed that PM fits into three hydrophobic binding pocket of TTR, one of the molecular targets of thyroid hormone disrupting chemicals (THDCs), and forms strong van der Waals interactions with six resides of TTR, including Leu8, Leu 101, Leu125, Thr214, Leu218 and Val229, thus altering TTR activity. Both in vivo and in silico studies clearly disclosed that PM potentially disrupts the thyroid endocrine system in fish. This study provides a rapid and cost-effective approach for identifying THDCs and the underlying mechanisms.

In Silico Approach To Identify Potential Thyroid Hormone Disruptors among Currently Known Dust Contaminants and Their Metabolites.

Thyroid hormone disrupting chemicals (THDCs) interfere with the thyroid hormone system and may induce multiple severe physiological disorders. Indoor dust ingestion is a major route of THDCs exposure in humans, and one of the molecular targets of these chemicals is the hormone transporter transthyretin (TTR). To virtually screen indoor dust contaminants and their metabolites for THDCs targeting TTR, we developed a quantitative structure-activity relationship (QSAR) classification model. The QSAR model was applied to an in-house database including 485 organic dust contaminants reported from literature data and their 433 in silico derived metabolites. The model predicted 37 (7.6%) dust contaminants and 230 (53.1%) metabolites as potential TTR binders. Four new THDCs were identified after testing 23 selected parent dust contaminants in a radio-ligand TTR binding assay; 2,2',4,4'-tetrahydroxybenzophenone, perfluoroheptanesulfonic acid, 3,5,6-trichloro-2-pyridinol, and 2,4,5-trichlorophenoxyacetic acid. These chemicals competitively bind to TTR with 50% inhibition (IC50) values at or below 10 μM. Molecular docking studies suggested that these THDCs interacted similarly with TTR via the residue Ser117A, but their binding poses were dissimilar to the endogenous ligand T4. This study identified new THDCs using an in silico approach in combination with bioassay testing and highlighted the importance of metabolic activation for TTR binding.

Cell type-dependent agonist/antagonist activities of polybrominated diphenyl ethers

There have been many concerns expressed regarding the possible adverse effects of thyroid hormone-disrupting chemicals including polychlorinated biphenyls and polybrominated diphenyl ethers (PBDEs), since thyroid hormones play crucial roles in normal vertebrate development. A vast amount of PBDEs have been used as flame retardants for the last two decades and our environment has been contaminated with them. Some PBDEs, especially hydroxylated PBDEs, reportedly show an affinity to the thyroid hormone receptor (TR) and act as thyroid hormone agonists, but in other studies they were reported to inhibit the actions of thyroid hormones. Therefore, in the present study, we investigated the binding affinities of PBDEs and their metabolites to TR and their ability to induce thyroid hormone-responsive transcription using luciferase reporter gene assays in two different cell lines, a pituitary cell line, MtT/E-2, and Chinese hamster ovary (CHO) cells. The binding assay showed that many of the examined PBDEs have significant affinity to TR. Interestingly, some of these PBDEs, such as 4′-OH-BDE-17 and 2′-OH-BDE-28, acted as agonists in the reporter gene assay in MtT/E-2 cells, while they acted as antagonists in CHO cells. Our results demonstrated that whether PBDEs and their metabolites are TR agonists or antagonists depends on the cell type used in the assay, which may suggest that the thyroid hormone-disrupting actions of PBDEs differ among target tissues or species.

Endocrine Disrupting Compounds – do we need to consider thyroid hormones? Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals

The thyroid hormone (TH) system is involved in many important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility, cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDC). Information on potential of chemicals causing THD is typically derived from animal studies. However, for most chemicals, this information is often limited or even unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern ‘-omics’ technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science [1] for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel recommends a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD.

An in vitro method to study the effects of thyroid hormone-disrupting chemicals on neuronal development

Thyroid hormones (THs) play critical roles for normal cerebellar development. It has been reported that several environmental chemicals may affect cerebellar development through TH system. One such example is the suppression of TH receptor (TR)-mediated transcription by polybrominated diphenyl ethers (PBDEs). To determine the effect of these chemicals on brain development, we established a primary culture system of rat cerebellar Purkinje cells. Using this system, as low as 10−10M TH induced Purkinje cell dendritic arborization and such effect was dose-dependent. We examined the effect of decabromodiphenyl ether (BDE209) using this system. Dendritic development of the Purkinje cells was suppressed by 10−10M BDE209, that was compatible to the result of the suppression of TR-mediated transcription by using reporter gene assay. These results suggest that TH plays a pivotal role in the development of the Purkinje cell dendrites. Together with in vitro assay system such as reporter gene assay and liquid chemiluminescent DNA-pull down assay, an in vitro protein–DNA binding assay, these assay systems provide us with precise information about environmental chemicals on brain development.

Application of metamorphosis assay to a native Japanese amphibian species, Rana rugosa, for assessing effects of thyroid system affecting chemicals

The aims of this study were to assess the utility of a metamorphosis assay for detecting thyroid hormone-disrupting chemicals using Rana rugosa, a domestic frog species in Japan, and to compare species differences in sensitivity to thyroxine (T 4) and propylthiouracil (PTU) among R. rugosa, Xenopus laevis and Xenopus ( Silurana) tropicalis. Tadpoles of R. rugosa (TK stages III/IV) were exposed to standard test chemicals for acceleration (T 4) and inhibition (PTU) of metamorphosis for 28 days in semi-static condition and total body length and developmental stage (TK stage) were recorded every week. T 4 (0.61 and 2.24 μg/L in actual concentrations) and PTU (19.73, 76.83, and 155.67 mg/L in actual concentrations) induced significant acceleration and inhibition of metamorphosis, respectively. The present results indicate that the metamorphosis assay is successfully applied to the domestic frog species, R. rugosa, suggesting this assay can be used for the assessment of chemicals on ecological impacts in wild frog species.